An Exon Trapping System Providing Size Selection of Spliced Clones and Facilitating Direct Cloning
Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have developed an exon trapping procedure based on the use of vector pETVSD2. Cosmid DNA is partially digested and cloned in pETV-SD2. DNA of an entire library of subclones is introduced into COS-1 cells and transiently expressed. RNA is isolated and vector-derived transcripts are amplified by RT-PCR. Cloning of the RT-PCR products, which contain a ColEI-origin of replication and a supF marker, is established by NotI digestion and intramolecular circularisation. Due to their shorter length, spliced clones are preferentially amplified and cloned.
Using this approach, we have been able to trap several exons from test cosmids of the DMD-gene, including the single 176 by exon 45 present in 40 kb of intronic sequences. We have applied this system in the search for coding sequences in various genomic regions, including the candidate region for the gene involved in facioscapulohumeral muscular dystrophy (FSHD) on 4q35. We have developed a screening procedure which allows more efficient identification of potential exon containing clones, thus discarding the high background of false positives.
KeywordsDuchenne Muscular Dystrophy Duchenne Muscular Dystrophy Splice Donor Splice Acceptor Yeast Artificial Chromosome
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